This project proposes to examine stimulus-response coupling in the human neutrophil. The basic hypothesis is that mobilization of Ca++ is an early event following membrane perturbation which then results in two sequellae: the mobilization and subsequent metabolism of arachidonic acid and activation of the respiratory burst. The role of Ca++ will be investigated by comparing Ca++ transport induced by various stimuli with arachidonate metabolism and respiratory burst activity. Specificity will be examined by the use of calcium channel blockers and the intracellular calcium chelator, TMB-8. The possible role of calmodulin in these processes will be evaluated. Lipid metabolism in the stimulated neutrophil will be examined using cells pre-labelled with 3H-labelled arachidonate and 14C-stearate. Emphasis will be placed on the source and mechanism of arachidonate release as well as its subsequent metabolism to various bioactive intermediates. The possible relationship of the lipid alterations to the respiratory burst will be examined by investigating the two phenomena in patients with chronic granulomatous disease as well as by incubating normal cells with various phospholipases and products of phospholipase metabolism. Finally, we are hypothesizing that the respiratory burst is mediated by a multi-component electron transport chain. Neutrophils (both resting and activated) will be fractionated by previously defined techniques and oxidase activity in each fraction (plasma membrane, specific granule, and azurophil granule) determined by three separate assays (O2- production, H2O2 production, and NADP formation). These activities will be correlated with the subcellular distribution of cytochrome b and ubiquinone. The kinetics of appearance of each of the above activities in the phagocytic vacuole will be measured. These experiments will be performed with both cells from normal donors and from patients with chronic granulomatous disease. Finally, reconstitution experiments will be performed in an attempt to activate the system in vitro by combining fractions containing the differing activities.